We propose to assess the robustness of a high-throughput mass spectrometric technology platform for quantitative measurement of multiple candidate biomarker proteins in complex samples such as human plasma. Specific assays will be developed for 300 candidate biomarker proteins drawn from published literature, pathway analysis, microarray, and proteomics discovery efforts. These assays will be used to measure biomarker levels in 200 selected cancer and 200 control plasma samples at three different laboratory sites using two variant MS platforms. The results will characterize the quantitative reproducibility of the assay methodology within- and between-laboratories, and over time. A series of standardized reagents will be developed allowing the assays to be implemented in other laboratories using similar standardized instrumentation. Our measurement platform makes use of well-established quantitative MS techniques used in the routine measurement of small molecules - so-called "multiple reaction monitoring" (or MRM) assays and stable isotope labeled internal standards for quantitation, here in the form of synthetic, labeled peptides. By measuring both the labeled and unlabeled (sample-derived) peptides by MS, the method provides a quantitative measure of the relative amounts of the signature peptide and therefore the protein that is derived from. In order to access lower abundance biomarkers (those in the lower 5 orders of magnitude of the overall 10 order plasma abundance scale) we will employ specific enrichment techniques using immobilized anti-peptide antibodies. After extensive optimization and characterization, our assays will be deployed on clinical plasma samples from breast cancer cases and controls. Key outcomes of this project will be to 1) demonstrate that we can make sensitive and specific assays quickly and inexpensively; 2) that the assays can be highly multiplexed, greatly reducing the cost-per-analyte; and 3) that the protocols and technology can be standardized and distributed. Several members of our CRTAC team are currently involved in proteomic consortia aimed at MS-based discovery of candidate breast cancer biomarkers in human samples and mouse models. These efforts are generating robust datasets that will also be leveraged by this CPTAC team to inform selection of candidates for assay development and ultimate validation.